We have employed the protein 4.1R gene as a model to study the molecular regulation of splicing during erythropoiesis. Protein 4.1R is a key element of the erythrocyte cytoskeleton. Two tightly regulated splicing events alter its expression and function during erythropoiesis: 1) An early event dictates exon 2'exclusion, which omits an upstream translation initiation site and produces only the "small" (80 kD) isoform. 2) A late event induces the inclusion of exon 16, which encodes a peptide critical for spectrin-actin binding. We have made progress toward understanding both events, including: 1) Identification of a stage-specific mFox-2A isoform that is up-regulated in late erythroid cells and critical for the exon 16 splicing switch;2) Demonstration that increased SF2/ASF expression in late erythroid cells stimulates exon 16 splicing by binding an exonic splicing enhancer;3) Quantification of a panel of splicing factors during erythroid differentiation, showing 4.1R splicing is modulated by varying the amount of selected ubiquitous and cell-type specific activators and inhibitors;two of these, PTB and a 40 kD novel protein are objects of the proposed studies;4) Identification of a coupled transcription and splicing pathway that regulates exon 2'splicing;and 5) Detection of a switch in promoter usage that results in the production of the 80 kD isoform in mature red cells. We now propose to define more precisely the key elements and mechanisms governing these two splicing events, and to establish the physiological role of the mFox-2A isoform that we discovered, employing the following specific aims: 1) To complete the characterization of the functional roles of PTB and a 40 kD protein in exon 16 splicing regulation and to characterize the mechanisms by which they promote exon 16 splicing activation. This will employ combined genetic and biochemical approaches in both in vitro and in vivo systems. 2) To determine the physiological impact of Fox-2A in directing erythroid specific alternative exon selection. The impact of Fox-2A will be analyzed by comprehensive analysis of erythroid specific splicing changes that occur in late cells, using SpliceArray technologies. 3) To investigate how transcription from the alternative exon 1A and 1C promoters governs exon 2'/2 splice site selection. These studies will provide insights into how particular factors function to program the differentiation of erythroid cells. PUBLIC HEALTH RELEVANCE: Many different forms of a protein can be generated from a gene by alternative mRNA splicing;improper production of these protein forms could have a profound impact on human diseases. We study a major example (Protein 4.1R) of how this splicing is regulated.